JP2019089769A - Protein tag, tagged protein and protein purification method - Google Patents

Abstract

To provide standard peptides for mass spectrometry for proteins to be subjected to mass spectrometry which makes it possible to easily recover proteins in high yield, and in which technology that enables comprehensive protein purification has been applied.SOLUTION: Provided is a standard peptide for mass spectrometry of a target protein that is a peptide mixture comprising a tagged peptide obtained by trypsinizing a fusion protein of a target protein of mass spectrometry and a protein tag consisting of a particular amino acid sequence derived from MafG protein.SELECTED DRAWING: None

Description

  The present invention relates to protein tags, tagged proteins, protein purification methods and the like. More specifically, the present invention relates to a protein tag or the like that can be conveniently recovered by insolubilizing a recombinant protein.

  Conventionally, purification of a recombinant protein is carried out by expressing the protein of interest as a fusion protein with an affinity tag, and utilizing the specific affinity which the affinity tag exhibits with other molecules, the fusion protein can be isolated from the soluble fraction It is done by isolation. The affinity tag also functions to enhance the solubility of the target protein and to solubilize and express the fusion protein.

  As affinity tags, His tag (histidine tag), GST tag (glutathione-S-transferase tag), MBP tag (maltose binding protein tag), FLAG tag, etc. are widely used. The His tag is a peptide containing about 6 to 10 histidine residues, and specifically binds to a metal ion such as nickel. GST tag and MBP tag specifically bind to low molecular weight compounds such as glutathione and maltose, respectively.

  For example, a nickel ion-immobilized chelate resin is allowed to flow through and adsorb a solution of a His-tagged protein. Subsequently, when the resin is allowed to flow through nickel ion or a low molecular weight compound that binds to nickel ion such as imidazole, the adsorbed protein can be eluted and recovered.

  The FLAG tag is a tag using an antigen-antibody reaction (epitope tag), and a FLAG tag-tagged protein can be isolated from the soluble fraction by binding to an anti-FLAG antibody. Other epitope tags include myc tag and HA tag, and the above-mentioned His tag and GST tag can also be used as epitope tags.

  In these affinity tags, it is also designed to cleave the fusion protein into a target protein and a tag by protease treatment so that the tag can be separated from the target protein. For example, if a fusion protein adsorbed to a resin through a tag is treated with a protease and cleaved between the target protein and the tag, only the target protein can be released from the resin and recovered.

  Patent Document 1 discloses a technique for specifically reacting a protein labeled with a composite tag consisting of a His tag and a FLAG tag with a nickel binding carrier and an anti-FLAG antibody binding carrier to separate and purify the protein. In addition, Patent Document 2 describes a method of recovering a target protein by separating a tag from a fusion protein by protease treatment using a peptide chain containing a cellulose binding region of a cellulolytic enzyme as an affinity tag.

Unexamined-Japanese-Patent No. 2003-292500 Japanese Patent Laid-Open No. 6-277088

Goshima, N., et al., Nature Methods. 2008, Vol. 5, No. 12, p. 1011-1017 "Human protein factory for converting the transcriptome into an in vitro expressed proteine." "Molecular Cloning, SECOND EDITION", 1989, 17.37-17.41, Cold Spring Harbor Laboratory Press "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", 1990, Vol. 2 UNIT 16.5-16.5.4 CURRENT PROTOCOLS

  Since conventional protein purification using affinity tag isolates the fusion protein from the soluble fraction using the specific affinity of the affinity tag, the fusion protein is expressed in a soluble state and in the soluble fraction Need to be fractionated. In addition, the affinity tag in the fusion protein needs to maintain specific affinity with other molecules.

  However, depending on the target protein, it is difficult to solubilize, and there is a large difference in solubilization rate among the proteins. Also, in some cases, the affinity tag in the fusion protein can not exert sufficient affinity with other molecules under the influence of the target protein.

  Therefore, in protein purification using conventional affinity tags, the recovery rate of the protein depends on the degree of solubility (solubilization rate) of the fusion protein and the degree of affinity of the affinity tag (affinity maintenance rate), and the recovery rate is It could be very low. Moreover, in the conventional purification method, it has been difficult to comprehensively synthesize and purify a large number of proteins regardless of their solubilization rate and affinity maintenance rate.

Then, this invention makes it possible to collect | recover a protein in high yield simply, and makes it a main object to provide the technique which enables comprehensive protein purification.
Another object of the present invention is to provide a standard peptide for mass spectrometry of a protein to be subjected to mass spectrometry, to which the present technology is applied.

  When expressing a recombinant protein, there are a protein that becomes soluble and a protein that becomes insoluble depending on the type of protein to be expressed. In addition, even one type of protein may be separated into two fractions, soluble and insoluble.

  As a result of intensive investigations, the present inventors have found an insolubilization tag that makes it possible to insolubilize the expressed protein regardless of the type of protein and to uniformly purify and recover the expressed protein from the insoluble fraction. . In addition, it has been found that the insolubilizing tag according to the present invention can be resolubilized at a lower concentration than the general concentration of the surfactant contained in the solvent for solubilizing the protein of the inclusion body. .

In order to solve the above problems, the present invention provides a protein tag comprising the full length or a part of the amino acid sequence of a MafG protein, or an amino acid sequence in which an amino acid to be a cleavage site by protease is inserted.
This protein tag can impart high insolubility derived from MafG protein to the protein to be tagged.
In this protein tag, the amino acid that becomes the cleavage site by protease can be arginine, which is the cleavage site of trypsin.
Specifically, this protein tag can comprise the amino acid sequence set forth in SEQ ID NO: 1-4.
The present invention also provides a protein tagged with a protein tag, which comprises the full-length or partial amino acid sequence of the MafG protein, or an amino acid sequence in which an amino acid to be cleaved by protease is inserted into the amino acid sequence. Do.
This tagged protein can be applied, for example, as a protein array in which proteins are immobilized on a support.
Furthermore, the present invention provides a peptide obtained by protease treatment of a protein tagged with a protein tag comprising an amino acid sequence in which an amino acid serving as a cleavage site for protease is inserted into the full-length or partial amino acid sequence of MafG protein. Also provide.
Since a peptide obtained by protease treatment of a protein tagged with a protein tag comprising an amino acid sequence having an amino acid sequence into which a protease cleavage site is inserted can be designed so that the tag-derived peptide becomes very short, It is suitable as a standard peptide for mass spectrometry of a protein to be subjected to mass spectrometry.

  The present invention also provides a vector that expresses the above-mentioned protein tag. This vector is intended to express a fusion protein of a target protein to be tagged and a protein tag.

Furthermore, according to the present invention, a protein of interest is tagged with a protein tag comprising the full-length or partial amino acid sequence of a MafG protein, or an amino acid sequence in which an amino acid to be cleaved by protease is inserted into the amino acid sequence. A protein purification method is provided which comprises a fusion protein preparation procedure and a purification procedure for recovering the tagged target protein into an insoluble fraction.
According to the above-mentioned protein tag, the high insolubility derived from the MafG protein can be imparted to the target protein, and the fusion protein can be insolubilized, so that the fusion protein can be recovered in a high yield in the insoluble fraction.
In this protein purification method, the fusion protein preparation procedure comprises a vector expressing a fusion protein of the target protein and the protein tag in a cell-free protein synthesis system (eg, wheat cell-free protein synthesis system) or a cell protein synthesis system. The procedure is used to synthesize the fusion protein. In addition, the purification procedure is a procedure of centrifuging the tagged target protein.

  In addition, the present invention relates to a method for purifying an antibody against a protein, wherein said protein and the entire or partial amino acid sequence of MafG protein, or an amino acid in which an amino acid serving as a cleavage site by protease is inserted into said amino acid sequence. There is also provided an antibody purification method characterized by using a fusion protein with a protein tag comprising the sequence.

  According to the present invention, a technology is provided that enables protein recovery with high yield and convenience, and enables comprehensive protein purification.

It is a figure explaining the protein tag (insolubilization tag) and tagged protein which concern on this invention. It is a figure explaining the protease cleavage site of insolubilizing tag and tagged protein. It is a figure explaining design of the protease cleavage site in insolubilization tag. It is a figure explaining the basic sequence of the vector concerning the present invention. It is a figure explaining an example of a procedure of a protein purification method concerning the present invention. It is a figure explaining the measurement principle of multiple reaction monitoring (MRM: multiple reaction monitoring). It is a figure explaining an example of a procedure of the antibody purification method concerning the present invention. It is a drawing substitute photograph which shows the result of having purified a signal transduction protein using an insolubilization tag (Example 1). It is a drawing-substituting graph which shows the result of having evaluated the fluorescence property of the fusion protein of an insolubilizing tag and a fluorescent protein (Example 2). It is a drawing-substituting graph which shows the result of having evaluated the enzyme activity of the fusion protein of an insolubilizing tag and a dephosphorylation enzyme (Example 2). It is a drawing-substituting graph which shows the result of having evaluated the enzyme activity of the fusion protein of an insolubilizing tag and a phosphorylation enzyme (Example 2). It is a drawing substitute graph which shows the result of having performed comprehensive protein purification using the insolubilization tag (Example 3). It is a drawing substitute photograph which shows the detection result of the autoantibody by the protein array produced in Example 4 (Example 5). It is a drawing substitute photograph which shows the detection result of the autoantibody by the protein array produced in Example 4 (Example 5). It is a drawing substitute graph which shows the detection result of the autoantibody by the protein array produced in Example 4 (Example 5). It is a drawing substitute graph which shows the detection result of the autoantibody by the protein array produced in Example 4 (Example 5). It is a figure explaining the vector used by E. coli cell expression system (Example 6). It is a drawing substitute photograph which shows the result of having refine | purified the protein using the insolubilization tag (Example 6). It is a figure explaining the domain of insolubilization tag (experiment example 1). It is a drawing substitute photograph which shows the result of having domainized the insolubilization tag (experiment example 1). It is a drawing-substituting photograph explaining the protein purification result using the insolubilizing tag which inserted the cleavage site of protease (Test Example 2).

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. The embodiment described below shows an example of a representative embodiment of the present invention, and the scope of the present invention is not narrowly interpreted. The description will be made in the following order.

1. Protein tag (1-1) Insolubilization tag (1-2) Design of cleavage site by protease Protein Purification Method (2-1) Fusion Protein Preparation Procedure (2-1-1) Vector (2-1-2) Expression System (2-2) Purification Procedure 3. Tagged protein and its application (3-1) Tagged protein (3-2) Standard peptide for mass spectrometry (3-3) antibody purification

1. Protein Tag (1-1) Insolubilization Tag The present inventors exhaustively searched for proteins showing high insolubility using a cell-free expression system (wheat cell-free system). As a result, we found MafG protein. It was revealed that the MafG protein does not show any solubility even when it is expressed as a fusion protein with a conventionally used solubilizing tag such as His tag or GST tag. The MafG protein is one of transcription factors and consists of an amino acid sequence of 162 residues (SEQ ID NO: 1). The base sequence of the coding region of the MafG gene is shown in SEQ ID NO: 5.

  The protein tag according to the present invention comprises an amino acid sequence of the full length or a part of the amino acid sequence of the above-mentioned MafG protein. The protein tag comprising the full-length or partial amino acid sequence of the amino acid sequence of the MafG protein is one that imparts high insolubility to the tagged protein derived from the MafG protein. That is, the protein tag according to the present invention functions as an “insolubilization tag” that is tagged to a soluble protein and insolubilized as a whole tagged protein (see FIG. 1). The present inventors maintain the enzyme activity or fluorescence of the fusion protein even when the protein is expressed as a fusion protein with this insolubilizing tag, with respect to various enzymes such as kinases and phosphatases and fluorescent proteins. (See Example 2).

  The amino acid sequence of the MafG protein contained in the insolubilization tag may be the entire or partial sequence of the amino acid sequence of the MafG protein, as long as the high insolubility of the MafG protein can be maintained. In the case of using a partial sequence, the partial sequence may be any portion of the entire length of the amino acid sequence of the MafG protein, and the number of amino acid residues in the partial sequence is not particularly limited. Furthermore, as long as the insolubilization tag can maintain the high insolubility exhibited by the MafG protein, in addition to the full-length or partial amino acid sequence of the amino acid sequence of the MafG protein, the amino acid sequence of any number of residues at its N-terminus or C-terminus You may have.

  In addition, the amino acid sequence of the MafG protein contained in the insolubilization tag is not limited to the amino acid sequence (SEQ ID NO: 1) of human MafG protein, and may be the amino acid sequence of other species homologue including mouse and rat.

  As a specific amino acid sequence of the insolubilization tag, for example, the amino acid sequence shown in SEQ ID NO: 1 can be mentioned. This amino acid sequence is identical to the full length amino acid sequence of human MafG protein. In addition, the amino acid sequence of the insolubilizing tag may be an amino acid sequence in which one or more amino acids are added to the N terminus or C terminus of the amino acid sequence shown in SEQ ID NO: 1.

  Furthermore, the specific amino acid sequence of the insolubilizing tag may be, for example, the amino acid sequence shown in SEQ ID NO: 2. This amino acid sequence is a partial sequence of human MafG protein and corresponds to residues 56 to 162 from the full N-terminus. The present inventors have found that a portion of residues 56 to 162 from the N-terminus of the human MafG protein contributes to insolubility (see Test Example 1). In addition, the amino acid sequence of the insolubilizing tag may be an amino acid sequence in which one or more amino acids are added to the N terminus or C terminus of the amino acid sequence shown in SEQ ID NO: 2.

(1-2) Design of Cleavage Site by Protease The insolubilization tag according to the present invention is a full-length or partial amino acid sequence of the MafG protein in order to apply the tagged protein to preparation of a standard peptide for mass spectrometry described later. It is preferable that the amino acid sequence comprises an amino acid sequence in which an amino acid to be a cleavage site by a protease is inserted. FIG. 2 is a schematic view showing an insolubilizing tag and a protease cleavage site of a tagged protein. The designed cleavage site in the amino acid sequence of the insolubilizing tag is shown by a dotted line, and the cleavage site inherent in the amino acid sequence of the target protein is shown by a dashed dotted line. Cleavage sites of the insolubilizing tag shown by dotted lines include both cleavage sites inherent to the MafG protein and cleavage sites newly provided.

  The amino acids to be inserted can be appropriately selected according to the type of protease. For example, in the case of trypsin having a cleavage specificity on the carboxyl side of lysine and arginine, lysine or arginine is inserted. As a protease, in addition to trypsin, LysC, GluC, AspN, chymotrypsin, V8 and the like can be used.

  The insertion position of the amino acid serving as a cleavage site by the protease inserted into the full length or a part of the amino acid sequence of the MafG protein is not particularly limited, but the length of the tag-derived peptide obtained after protease treatment is 6 residues or less It is preferable to be inserted in such a position that In addition, the number of insertions of amino acids to be cleavage sites is not particularly limited.

  When the protease is trypsin, a suitable amino acid sequence of the insolubilization tag is the amino acid sequence shown in SEQ ID NO: 3 (see FIG. 3). The amino acid sequence shown in SEQ ID NO: 3 is a sequence obtained by inserting 10 residues of arginine into the amino acid sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 2 has 6 amino acid residues between arginine and arginine. It is a sequence modified to be less than a group. In the amino acid sequence shown in FIG. 3, the underlined "R" indicates an arginine internal to wild type (Wild-type) MafG protein, and the arrowed "R" indicates an inserted arginine (10) Indicates

  Furthermore, the amino acid sequence shown in SEQ ID NO: 4 is a sequence obtained by inserting 17 residues of arginine into the amino acid sequence shown in SEQ ID NO: 2, and compared to the amino acid sequence shown in SEQ ID NO: 3, the amino acid between arginine and arginine It is a sequence modified to reduce the number further. In the amino acid sequence shown in FIG. 3, “R” with an arrow indicates arginine (7) further inserted into the amino acid sequence shown in SEQ ID NO: 3.

2. Protein Purification Method (2-1) Fusion Protein Preparation Procedure A protein purification method using the above-described insolubilizing tag is described. Protein purification methods include fusion protein preparation procedures and purification procedures.

  In the fusion protein preparation procedure, a target protein to be purified is prepared as a fusion protein with an insolubilizing tag. The fusion protein can be obtained by introducing a vector capable of expressing the target protein as a fusion protein with the insolubilization tag into an expression system by connecting the DNA sequence encoding the target protein to the DNA sequence encoding the insolubilization tag. .

(2-1-1) Vector FIG. 4 shows an example of the basic sequence of the vector. The vector shown in the figure is a DNA sequence (tag sequence) encoding the amino acid sequence of the insolubilization tag upstream (5 'side) of the position (open reading frame (ORFs)) where the cDNA encoding the amino acid sequence of the target protein is inserted. ) Is arranged. Further upstream of the tag sequence, a transcription promoter sequence and a translation enhancer sequence are located. The transcriptional promoter sequence and the translational enhancer sequence are exemplified herein as the SP6 promoter sequence and the omega sequence often used in the wheat cell-free protein synthesis system, respectively, but are not limited thereto. The tag sequence can also be linked downstream (3 'side) of the ORFs. That is, the insolubilizing tag can be used as an N-terminal tag or a C-terminal tag.

  A sequence for gene recombination may be provided before and after the tag sequence and the ORFs. Here, a restriction enzyme XhoI site is provided upstream of the tag sequence, a KpnI site downstream, and an attB1 site upstream of the ORFs, and an attB2 downstream.

(2-1-2) Expression System A cell-free system or a cell system can be used as the expression system, and in particular, a wheat cell-free expression system can be used. Other cell-free protein synthesis systems include E. coli, insects, rabbit reticulocytes and the like. Examples of cell-based protein synthesis systems include E. coli, mammalian cells, insect cells, yeast and the like.

  In the expression of a fusion protein in a cell-free system, first, a DNA sequence (tagged cDNA) encoding a target protein and a protein tag is amplified from the above vector by a nucleic acid amplification method such as PCR. Next, RNA (tagged RNA) is transcribed by in vitro transcription from tagged cDNA. Then, in vitro translation is performed using the tagged RNA as a mixture with the wheat cell extract (see FIG. 5).

  In addition, as a cell-based protein synthesis system, conventionally known systems using E. coli, insect cells and mammalian cells can be used. Gene transfer into cells can be carried out according to known transfection methods such as calcium phosphate method, electroporation method, lipofection method and microinjection method.

(2-2) Purification Procedure Next, in the purification procedure, the target protein expressed as a fusion protein with the insolubilizing tag is recovered in the insoluble fraction. The target protein tagged with the insolubilization tag is insolubilized as a whole tagged protein. Therefore, by centrifuging the mixed solution after in vitro translation in the cell-free expression system or the cell lysate in the cell expression system etc., the target protein can be easily pelleted into the insoluble fraction and recovered (FIG. 5) reference).

  Centrifugation can be carried out according to a routine method, and the conditions are, for example, 15,000 × g, 20 minutes, 4 ° C. In order to prevent the soluble protein from being mixed into the insoluble fraction as a contaminating protein, it is preferable to add a surfactant such as Tween 20 to the solution to be centrifuged.

  In addition, the target protein tagged with the insolubilizing tag can also be recovered to a filter impermeable fraction using a filter. The mixed solution after in vitro translation in the cell-free expression system or the cell lysate in the cell expression system is passed through a filter with a pore size of about 0.22 μm. Thereby, the soluble protein which permeate | transmits a filter and the target protein captured by a filter can be isolate | separated.

  The insolubilized tag according to the present invention can impart high insolubility derived from MafG protein to the target protein to be tagged, and even a highly soluble target protein can be insolubilized as a whole tagged protein . Therefore, according to the protein purification method using this insolubilization tag, unlike the conventional method using an affinity tag, the target protein can be purified with a high recovery rate without depending on the degree of solubility (solubilization rate).

  Further, in the protein purification method according to the present invention, the tagged target protein can be recovered by a simple technique such as centrifugation. Therefore, in this method, unlike the conventional method using affinity tags, the target protein can always be purified with a high recovery rate without concern about the degree of affinity (affinity maintenance rate) of the tags in the fusion protein.

  Furthermore, according to the protein purification method of the present invention, even a protein that has been difficult to purify because of the low solubilization rate or affinity retention rate by the conventional method can be purified. It is possible to comprehensively synthesize and purify regardless of the solubilization rate and the affinity maintenance rate.

  The recovered insolubilized tag protein can be treated with a surfactant, treated with a protein denaturant such as 7 M guanidine hydrochloride or 7 M urea, and solubilized with an acid or an alkaline solution. Furthermore, the tagged protein of the present invention can be solubilized in the presence of sodium dodecyl sulfate (SDS) at a concentration of 0.04 to 1% (w / v) (see Test Examples 3 and 4).

  Generally, for resolubilization of insolubilized proteins, use of a chaotropic salt with high denaturation power such as guanidine hydrochloride (Non-patent document 2, Non-patent document 3) or about 2% SDS for SDS-PAGE. In many cases, a so-called SDS buffer to which a reducing agent is added is used. However, in a solvent containing a chaotropic salt or surfactant at a high concentration, the target protein may be denatured, for example, if the target protein is an enzyme, the enzyme activity may not be maintained. In addition, in order to use a protein solubilized in a solvent containing a high concentration of chaotropic salt or surfactant at a biochemical assay or the like by adding an enzyme such as a protease, dilution, dialysis, or the like can be used. It is necessary to lower the concentration of the chaotropic salt or surfactant in the solvent by membrane filtration or the like.

  On the other hand, the tagged protein according to the present invention can be solubilized, for example, at a concentration of 0.04 to 1% (w / v) of SDS. Therefore, the tagged protein recovered in the insolubilized fraction can be used in the next procedure without performing complicated operations such as dilution, dialysis, and ultrafiltration. The next procedure is, for example, a protease treatment procedure in using tagged proteins as a standard peptide for mass spectrometry, which will be described later, and a procedure for binding tagged proteins to a protein array substrate.

3. Tagged Protein and its Application (3-1) Tagged Protein The tagged protein according to the present invention is characterized by being obtained by the above-described protein purification method and tagged with an insolubilizing tag. Depending on the structure of the vector used, the tagged protein has an insolubilization tag attached to at least one of the N-terminus and C-terminus of the target protein.

  In addition, as described above, the tagged protein according to the present invention can be solubilized from the insoluble state. Therefore, according to the present invention, a protein of interest is tagged with a protein tag comprising a full-length or partial amino acid sequence of a MafG protein, or an amino acid sequence in which an amino acid serving as a cleavage site by protease is inserted into the amino acid sequence. Fusion protein preparation procedure, purification procedure for recovering the tagged target protein in the insoluble fraction, and solubilization procedure for resolubilizing the tagged target protein recovered in the insoluble fraction in a solvent, It can also be a protein production method. The solvent is preferably one containing SDS at a concentration of 0.04 to 1% (w / v).

  According to the insolubilizing tag of the present invention, a large number of proteins can be synthesized and purified regardless of their solubilization rate and affinity maintenance rate. Therefore, a comprehensive protein library (in vitro proteome) can be prepared by inserting a generally available cDNA library into the ORFs of the vector to create an expression clone library, and performing protein expression and purification.

  Protein libraries prepared as in vitro proteomes can be applied to, for example, protein arrays and the like. The protein is solubilized using a surfactant such as sodium dodecyl sulfate (SDS), a denaturant such as guanidine hydrochloride or urea, or an acid or alkaline solution, and immobilized on a support such as nitrocellulose membrane or various array substrates. Create a protein array. Protein arrays can be used, for example, for applications such as screening of autoantibodies in serum.

(3-2) Standard peptide for mass spectrometry The tagged protein according to the present invention can be applied to preparation of a standard peptide for mass spectrometry (peptide for internal standard). In LC / MS / MS (Liquid chromatography / mass spectrometry / mass spectrometry), CE / MS / MS (capillary electrophoresis / mass spectrometry / mass spectrometry) and GC / MS / MS (gas chromatography / mass spectrometry / mass spectrometry), selection Analyzes referred to as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) have been performed. Since SRM and MRM have extremely high specificity, they are excellent in the quantification of ultra trace components.

  FIG. 6 shows a general measurement principle of MRM. In the MRM, first, specific ions (precursor ions) are selected in Q1 (first mass spectrometer) for various ions ionized by the ionization probe. Next, precursor ions are broken (collision induced dissociation) in a collision cell (Q2, collision chamber), and specific ions are detected from among the broken ions (product ions) in Q3 (second mass spectrometer).

  In MRM, it is possible to set a plurality of channels in one measurement, and it is possible to detect and quantify only a specific type of protein from among crude proteins (crude protein products). Protein quantification by MRM is performed by determining the absolute amount of the peptide to be quantified from the peak area ratio of the peptide to be quantified and the stable isotope labeled peptide as an internal standard and a calibration curve.

  For high-precision quantification, it is necessary to select in advance a peptide having high ionization efficiency as a peptide for internal standard among peptides generated from a protein to be quantified. The selection of the internal standard peptide is performed while actually measuring the protein to be quantified and confirming the MS / MS spectrum, so it is necessary to prepare the protein to be quantified which is purified for actual measurement.

  Further, the peptide for internal standard is labeled with a stable isotope so as to be distinguishable from the peptide derived from the protein to be quantified in crude protein purification (sample), mixed with the sample, and provided for measurement. For this reason, in MRM, it is necessary to prepare an internal standard peptide for each measurement.

  The tagged protein according to the present invention can be synthesized and purified for all proteins regardless of the solubilization rate of the protein and the affinity maintenance rate of the fusion protein. It can be suitably used as a protein and a protein for preparation of a peptide for internal standard. That is, the purified tagged protein is treated with protease and analyzed by LC / MS / MS, whereby the peptide for internal standard optimal for MRM can be selected. Moreover, the protease treatment can be performed in a state where the tag is added without removing the tag from the tagged protein in advance. Therefore, there is no need to remove the tag added for purification from the tagged protein after purification. Furthermore, a peptide mixture obtained by protease treatment of a tagged protein labeled with a stable isotope can be used as it is as an MRM internal standard peptide.

  When the tagged protein according to the present invention is used for a peptide for internal standard such as MRM, the number of amino acid residues constituting the protein tag is preferably smaller. This is because as the number of amino acid residues constituting the protein tag increases, the peptide contained in the protease digestion product tends to be longer, and the peptide detected in mass spectrometry tends to interfere with the measurement of the target protein. Therefore, in the amino acid sequence of the MafG protein, it is preferable to use a portion containing SEQ ID NO: 2 involved in insolubility as a protein tag. By using the amino acid sequence shown in SEQ ID NO: 2 as a tag, the number of amino acid residues in the protein tag can be reduced and the insolubility of the protein tag can be maintained.

  Furthermore, synthesis is carried out using an insolubilization tag comprising an amino acid sequence (see SEQ ID NOS: 3 and 4) in which an amino acid serving as a cleavage site for protease is inserted into the full length or a part of the amino acid sequence of the amino acid sequence of MafG protein. And purified tagged proteins are suitable for use.

  The amino acid to be a cleavage site by protease is inserted at such a position that the length of the peptide (tag-derived peptide) derived from the insolubilizing tag and obtained after the protease treatment is 6 residues or less (see FIG. 2). By setting the length of the insolubilized tag-derived peptide obtained after the protease treatment to be 6 residues or less, in the peptide for internal standard in which the tagged protein is protease-treated, the tag-derived peptide and the protein-derived peptide Clearly distinguishable. As a result, it is possible to prevent the tag-derived peptide from being erroneously selected as the internal standard peptide or preventing the tag-derived peptide from becoming noise during the MRM measurement.

  For example, when using trypsin as a protease, arginine or lysine is preferable as the amino acid inserted into the amino acid sequence of the protein tag according to the present invention. The length of the peptide derived from the protein tag can be further shortened by increasing arginine or lysine which is a cleavage site of trypsin in the amino acid sequence of the protein tag. On the other hand, when arginine and lysine which are hydrophilic amino acids are inserted into a protein tag showing insolubility, there is a possibility that the property of the protein tag may change.

  In particular, when the number of arginine residues inserted into the tag protein consisting of 107 amino acid residues shown in SEQ ID NO: 2 is less than 17, the insolubility of the tag protein is maintained and the length of the peptide derived from the tag protein is shortened. be able to. Therefore, a protein tag in which less than 17 arginine residues are inserted into the amino acid sequence shown in SEQ ID NO: 2 is suitable as a tag used to purify the peptide for internal standard in mass spectrometry. In particular, the number of arginine residues inserted into the amino acid sequence shown in SEQ ID NO: 2 is preferably 1 or more and 16 or less, and particularly preferably 6 or more and 10 or less.

(3-3) Antibody Purification The tagged protein according to the present invention can also be applied to the purification of antibodies.

  The procedure for antibody purification will be described with reference to FIG. First, an antibody solution containing an antibody to be purified (anti-protein T antibody) and the tagged protein T are mixed, and centrifuged (for example, 15,000 × g, 20 minutes, 4 ° C.). After centrifugation, the anti-protein T antibody is separated in the sediment as a complex bound to tagged protein T. Next, the anti-protein T antibody is dissociated from the complex using an antibody dissociation buffer and centrifuged again. By this centrifugation, the tagged protein T becomes a precipitate and moves to the insoluble fraction, so that the anti-protein T antibody can be recovered in the supernatant.

  Since the tagged protein according to the present invention can be synthesized and purified for all proteins regardless of the solubilization rate of the protein and the affinity maintenance rate of the fusion protein, purification of all anti-protein antibodies can be realized. In addition, although the example which isolate | separates a protein-antibody complex by centrifugation is shown here, the complex by trap by a filter etc. can also be isolate | separated. By treating the complex trapped by the filter with an antibody dissociation buffer, the anti-protein antibody dissociated from the complex can be eluted and recovered.

Example 1
1. Purification of signal transduction protein using insolubilization tag (wheat cell-free expression system)
In this example, a wheat cell-free expression system was used to synthesize and purify a fusion protein of an insolubilizing tag according to the present invention and a signal transduction protein.

A method described in Non-Patent Document 1 using an entry clone in which an open reading frame (ORF) sequence of a signal transduction protein is cloned, and a destination vector for insolubilizing tag fusion for a wheat cell-free expression system shown in FIG. According to, the protein synthesis using wheat germ extract (WEPRO7240, cell free science company) was performed. Accession numbers of signal transduction protein gene symbols (Gene symbol), public database (GenBank: http://www.ncbi.nlm.nih.gov/genbank/), and entry clone numbers (ID) “Table 1” ~ Shown in "Table 3".
The solution after synthesis (crude protein solution) was diluted 4-fold with PBS in order to reduce contaminating proteins contaminating the purification, and centrifuged at 15,000 × g for 20 minutes at 4 ° C. The supernatant fraction was removed, and the obtained sediment (insoluble fraction) was used as a purified protein fraction.
For comparison, protein synthesis was similarly performed using a GST tag fusion destination vector. The solution after synthesis was diluted 4-fold with PBS, adsorbed onto glutathione resin (GE Pharmacia), eluted with 0.8 M glutathione, and the purified protein was recovered.

  The results are shown in FIG. FIG. 8A shows the results of SDS-PAGE of a protein purified using a GST tag. FIG. 8B shows the results of SDS-PAGE performed on the protein purified using the insolubilizing tag. Unlike FIG. 8A, in FIG. 8B, bands can be confirmed in all proteins, and bands of each protein are observed to be deeper than in FIG. 8A. From these results, it was shown that, by using the insolubilizing tag, the protein can be purified at a high rate and at a high yield as compared with the conventional purification method using a GST tag.

Example 2
2. Purification of Fluorescent Protein or Enzyme Using Insolubilizing Tag In this example, the fusion protein of the insolubilizing tag according to the present invention and the fluorescent protein or enzyme is purified, and the purified fusion protein maintains its fluorescence or enzyme activity. Was confirmed.

  In the same manner as in Example 1, a fusion protein of a fluorescent protein and an insolubilizing tag was synthesized. As the fluorescent protein, mVenus (Venus A206K, GenBank accession No. DQ092360.1) was used. The result of measuring the fluorescence of the crude protein solution (excitation wavelength 515 nm, fluorescence wavelength 528 nm) is shown in FIG.

  Even in the fluorescent protein fused with the insolubilizing tag (N-terminal tag), fluorescence having half the intensity of the fluorescent protein not tagged was confirmed. In addition, fluorescence was also confirmed in a fluorescent protein in which an insolubilizing tag was fused to the C-terminus.

  Further, in the same manner as Example 1, a fusion protein of a dephosphorylation enzyme and a phosphorylation enzyme and an insolubilization tag was synthesized. As the phosphatase, DUSP3, PTPN1 and PTPN6 were used. In addition, tyrosine kinases WEE1 and Hck1 were used as phosphorylation enzymes. The accession numbers of public databases of each enzyme are shown in "Table 4".

  The phosphatase activity was measured by the following method. The crude protein solution was centrifuged as in Example 1 to obtain a purified protein fraction. For purified protein fractions, in order to increase the degree of purification, resuspension with buffer (50 mM Tris-HCl, pH 7.5) and centrifugation were repeated twice each to remove contaminating proteins. To the pellet after centrifugation, 3.0 μl of buffer solution (50 mM Tris-HCl, pH 7.5) was added and suspended by sonication. The phosphatase activity was measured by absorbance measurement according to a standard method using the suspension and the pNpp chromogenic substrate.

  The results are shown in FIG. For each of DUSP3, PTPN1 and PTPN6, the enzyme activity of the supernatant fraction obtained by centrifuging the crude protein solution is shown on the left of the graph, and the enzyme activity of the insoluble fraction (purified protein fraction) on the right of the graph. In any of the enzymes, it was confirmed that the fusion protein with the insolubilizing tag maintains the activity.

  The tyrosine kinase activity was measured by the following method. The purified protein fraction obtained by centrifuging the crude protein solution in the same manner as in Example 1 is dissolved in LDS sample buffer (Life Technologies), and electrophoresis and western blotting are carried out using the NuPAGE electrophoresis system (Life Technologies). The autophosphorylation ability of tyrosine kinase was evaluated. E-PLUS chemiluminescence detection kit using P-Tyr-100 antibody (mouse IgG, Cell Signaling) as the primary antibody and anti-mouse IgG antibody (sheep IgG, HRP label, GE Healthcare) as the secondary antibody Detection was performed using (GE Healthcare).

  The results are shown in FIG. Lane 1 is the crude protein fraction of untagged WEE1, lane 2 is the crude protein solution of insolubilized tag-fused WEE1, lane 3 is the centrifuged supernatant fraction of insolubilized tag-fused WEE1, lane 4 is the insolubilized tag-fused WEE1 It is a purified protein fraction of centrifuged sediment. Also, lane 5 is a crude protein fraction of Hck1 that is not tagged, lane 6 is a crude protein solution of insolubilized tag fusion Hck1, lane 7 is a supernatant fraction obtained by centrifugation of insolubilized tag fusion Hck1, and lane 8 is an insolubilized tag fusion Purified protein fraction of centrifuged sediment of Hck1. A band (see circle in the figure) binding to the anti-phosphorylated tironcin antibody (P-Tyr-100 antibody) is detected in lanes 4 and 8, and the fusion protein with the insolubilizing tag in any enzyme has autophosphorylation ability It was confirmed that the

  From the results of this example, it was confirmed that tagging of a protein with an insolubilizing tag does not affect the fluorescence and enzyme activity of the protein.

Example 3
3. Exhaustive Protein Purification Using Insolubilizing Tag In this example, exhaustive protein purification was performed using the insolubilizing tag according to the present invention.

  In the same manner as in Example 1, 1026 types of metabolic proteins registered in KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/kegg_ja.html) are combined with insolubilizing tags. The fusion protein was synthesized, recovered by centrifugation and collected in the sediment fraction for purification.

  The results of electrophoresis quantification of the purified protein by protein fluorescent prelabeling method are shown in FIG. The protein fluorescent prelabeling method was performed by introducing a fluorescent dye Cy5 into a lysine residue by amine coupling.

Example 4
4. Preparation of Protein Array In this example, a protein purified by using the insolubilizing tag according to the present invention was bound to a substrate to prepare a protein array.

  In the same manner as in Example 1, a fusion protein of a protein (see Table 5) and an insolubilization tag was synthesized using an entry clone in which the ORF sequence of the protein shown in Table 5 was cloned, and the precipitate was separated by centrifugation Min) was obtained as a purified protein fraction. The purified protein fraction is suspended in a solution (0.04% SDS (w / v), 0.1 M phosphate buffer (pH 7.8)), shaken for about 1 minute, and mixed for 1 minute. Sonication (high-frequency output 160 W, 40 kHz) three times to obtain a solubilized protein of the purified protein with an insolubilized tag.

  As a substrate of the protein array of this example, two types of substrates, Super NHS manufactured by Arrayit, and FAST Slide 1-Pad manufactured by GE Healthcare, were prepared. The lysate containing each fusion protein is spotted on each substrate using a microdispenser or pin tool, and the binding reaction between the fusion protein and the substrate surface is allowed to occur according to the protocol of the provider of each substrate. The In addition, in order to provide a negative control or a positive control in Example 5 described later, the fluorescent protein Venus and purified human IgG were also attached to the above substrate.

  For the substrate, after drying the lysate containing the attached protein, the substrate surface is prepared using TBST (20 mM Tris-HCl (pH 8.0), 134 mM NaCl, 0.1% (v / v) Tween 20). Were removed to remove proteins that were not bound to the substrate surface, and the protein array was completed.

Example 5
5. Detection of Autoantibodies by Protein Array The protein array prepared in Example 4 was used to detect autoantibodies in serum.

  Human serum containing anti-TRIM21 antibody and anti-CT45A5 antibody was prepared as autoantibodies. This human serum was diluted 1000-fold with TBST containing 3% (w / v) skimmed milk to make a primary antibody solution. The protein array prepared in Example 4 was previously immersed in TBST containing 3% (w / v) skimmed milk at room temperature for 1 hour to perform blocking of the substrate surface. After blocking, the surface of the substrate was immersed in the primary antibody solution and left at room temperature for 1 hour while shaking the primary antibody solution. Thereafter, the substrate surface was cleaned using TBST.

  In order to detect autoantibodies contained in the primary antibody solution, prepare an anti-human IgG antibody labeled with fluorescent dye Alexa 647 as a secondary antibody, and use this anti-human IgG antibody as a 3% (w / v) skim milk What was diluted 1000 times with TBST containing was made into the secondary antibody solution. The surface of the substrate was immersed in the secondary antibody solution, and the secondary antibody solution was placed at room temperature for 1 hour while shaking. Thereafter, the substrate surface was cleaned using TBST. After treatment with the secondary antibody, using a fluorescence scanner, the excitation wavelength is 635 nm, PMT is 420 V for Super NHS protein array, and 200 V for PMT for FAST Slide substrate, and the fluorescence emitted from the substrate surface is It was measured.

  The results of this example are shown in FIGS. FIG. 13 and FIG. 14 show the measurement results of the fluorescence emitted from the spot to which each protein is attached on the substrate. FIG. 13 shows the result of using SuperNHS as a substrate, and FIG. 14 shows the result of using FAST Slide 1-Pad as a substrate. Moreover, FIG. 15 is a graph which quantified fluorescence intensity of each spot shown in FIG. 13, and FIG. 16 is a graph which quantified fluorescence intensity of each spot shown in FIG. The spots indicated by arrows in FIG. 14 were excluded from the measurement targets of fluorescence because they were portions where the protein binding to the substrate was insufficient.

  As shown in FIG. 13 to FIG. 16, in the TRIM 21 and CT 45 A 5 spots in which autoantibodies are contained in serum, the fluorescence from the secondary antibody was measured. Also, the measured fluorescence intensity was proportional to the amount of protein attached to the substrate. This indicates that the primary antibody is specifically bound to the protein bound to the substrate. On the other hand, for the spots of MGLL, which is a protein containing no antibody in serum, the measured fluorescence intensity was lower than the fluorescence intensity measured with the above-mentioned TRIM 21 or CT 45 A 5 spots. Moreover, in the spot of the purified human IgG which is a positive control, the fluorescence intensity comparable to TRIM21 was measured. On the other hand, in the spot of Venus which is a negative control, the measured fluorescence intensity was comparable to the above-mentioned spot of MGLL.

  From the results of this example, it was confirmed that the protein purified using the insolubilizing tag according to the present invention is purified to an extent suitable for specific detection with an antibody in analysis using a protein array. Therefore, proteins purified using the insolubilizing tag according to the present invention can be used for protein arrays by binding them on a substrate.

Example 6
6. Purification of protein using insolubilization tag (E. coli intracellular expression system)
In this example, a fusion protein of the insolubilized tag according to the present invention and GST was purified using a colonic cell in-cell expression system.

In this example, an E. coli expression vector (pDEST15) shown in FIG. 17 was used. FIG. 17A is a vector for synthesizing GST to which an insolubilization tag has been added. On the other hand, FIG. 17B is a vector for synthesizing only GST. The vectors shown in FIGS. 17A and 17B were introduced into E. coli to obtain transformed E. coli. The transformed E. coli is cultured overnight at 37 ° C., and the resulting E. coli culture solution is diluted to an OD ₆₀₀ of 0.5, and then the concentration is 0.1% in this dilution. L-arabinose was added to initiate expression induction. Dilutions were collected immediately after induction of expression (0 hour) and 3 hours after induction of expression, and cells were collected by centrifugation.

  100 μl of PBS was added to the obtained cells, resuspended, and subjected to 20 times of 1 minute sonication (high frequency output 160 W, 40 kHz). After the sonication, a part of the suspension containing cells was used for SDS-PAGE as it was. Also, the suspension was centrifuged at 19000 × g for 20 minutes at 4 ° C. to obtain a supernatant and a sediment. These supernatants and sediments were also used for SDS-PAGE.

  The results of this example are shown in FIG. At 3 hours after expression, in the suspension obtained from E. coli into which the vector shown in FIG. 17A was introduced, a band was observed at the position of the molecular weight judged to be GST with the insolubilization tag added to the C end (Fig. 18A, T). On the other hand, in the suspension obtained from E. coli into which the vector shown in FIG. 17B was introduced, a band was observed at the position of molecular weight judged to be GST (see FIG. 18B, T).

  In addition, with respect to the supernatant obtained by centrifuging the suspension and the sedimentation, GST in which the insolubilization tag was added to the C end was almost entirely recognized in the sedimentation (see FIG. 18A, arrowhead). On the other hand, GST was almost entirely found in the supernatant (see FIG. 18B, arrowhead).

  From the results of this example, it was confirmed that the insolubilizing tag according to the present invention insolubilizes the fusion protein to which the insolubilizing tag is added, even when the protein is synthesized using an E. coli cell expression system.

<Test Example 1>
7. Domainization of Insolubilization Tag In this test example, a partial sequence (domain) that contributes particularly to insolubility among the full-length amino acids of the MafG protein was identified.

  The full length amino acid of MafG protein is divided into 3 domains, domain A is amino acid 1 to 55 from the N terminus, domain B is amino acid 56 to 109 from the N terminus, and domain C is 110 to 162 from the N terminus It was an amino acid (see FIG. 19). A vector capable of expressing five types of insolubilization tags consisting of the amino acid sequences of full-length, only domain A, domain A and domain B, only domain B, domain B and domain C, and only domain C was constructed. Methionine is added to the N terminus of each insolubilizing tag. In the ORFs of the vector, cDNA of fluorescent protein mVenus or GST which is a highly soluble protein was inserted. Fusion proteins were synthesized in a wheat cell-free system using these vectors, centrifuged, and subjected to SDS-PAGE.

  The result of having compared the recovery amount between five types of insolubilized tags is shown in FIG. In any of the fusion protein with Venus and the fusion protein with GST, the insolubilization tag (B + C) consisting of the amino acid sequences of domain B and domain C has a recovery amount similar to that of the insolubilization tag (MG) consisting of the full length amino acid sequence there were. On the other hand, in the insolubilizing tag consisting of the amino acid sequence of only domain A (A), domain A and domain B (A + B), only domain B (B), only domain C (C), the insolubilizing tag consisting of the full length amino acid sequence Recovery amount decreased compared to (MG). From these results, it was revealed that domain B and domain C among the total amino acid lengths of the MafG protein contribute to insolubility, and the insolubilization tag can function even if it is reduced to 107 residues of domain B and domain C.

Test Example 2
8. Insertion of Protease Cleavage Site In this test example, the amino acid sequence to be a cleavage site by protease was inserted into the amino acid sequence of domain B and domain C obtained in Test Example 1, and the amino acid sequence of the insolubilization tag was modified.

  The insolubilization tag consisting of the amino acid full-length (SEQ ID NO: 1) of MafG protein is Version 1, Version 2 (SEQ ID NO: 3) in which 10 arginines are inserted in the amino acid sequence of domain B and domain C, and 7 arginines are inserted in Version 2 An insolubilized tag of Version 3 (SEQ ID NO: 4) was prepared. The specific amino acid sequence of each insolubilization tag can be referred to FIG. The insolubilizing tag of Version 2 is one in which arginine is inserted and added such that the length of the tag-derived peptide obtained after trypsin treatment is 6 residues or less. The insolubilizing tag of Version 3 is one in which more arginine is inserted and added.

  Protein synthesis is performed in a wheat cell-free system using a vector capable of expressing a fusion protein of these insolubilization tag and 12 kinds of proteins (see Table 6), centrifuged, and subjected to SDS-PAGE to recover the fusion protein The amounts were compared.

(The lane numbers in the table correspond to the lane numbers in Figure 21. The public database Ensembl can refer to http://asia.ensembl.org/index.html)

  The results are shown in FIG. Fig. 21A shows the results of SDS-PAGE of a fusion protein with the insolubilizing tag of Version 1, Fig. 21B that of Version 2 and Fig. 21C that of Version 3. With the insolubilized tag of Version 2, a protein recovery rate equivalent to that of the insolubilized tag of Version 1 was obtained. On the other hand, in the insolubilized tag of Version 3, the protein recovery rate was slightly lower than that of the insolubilized tag of Version 1. From this result, when the shortest amino acid sequence (107 residues) domainized in Test Example 1 is used as the amino acid sequence of the insolubilization tag, the preferred number of insertions of amino acids to be cleavage sites by protease is less than 17 It was suggested. The more amino acids to be inserted, the shorter the length of the tag-derived peptide obtained after protease treatment, but if it is too much, the insolubilization tag's insolubility decreases and the fusion protein's solubility increases and the recovery efficiency by centrifugation decreases. It was suggested that there is a case.

<Test Example 3>
9. Examination of Resolubilization Conditions of Tagged Protein The appropriate solvent was examined for resolubilization of the fusion protein (tagged protein) to which the insolubilized tag was recovered as the insolubilized fraction (tagged protein).

  In the same manner as in Example 1, a fusion protein of the fluorescent protein mVenus (Venus A206K, GenBank accession No. DQ092360.1) and the insolubilizing tag was synthesized using a wheat cell-free expression system. Also, using the entry clone (entry clone ID FLJ40036AAAF) into which the ORF sequence of immunoglobulin heavy chain gamma 3 constant region (IgHG3, GenBank accession No. AK097355) has been cloned, the fusion protein of IgHG3 and the insolubilization tag is also used without wheat. It was synthesized using a cell expression system.

The suspension containing the synthesized tagged protein was centrifuged under the conditions of 19,000 × g, 20 minutes, 4 ° C. to collect the tagged protein as an insolubilized fraction. One of the solvents shown in Table 7 is added to the obtained insolubilized fraction, and after mixing with shaking for 1 minute, ultrasonication (high frequency output 160 W, 40 kHz) is performed 3 times for 1 minute. The The suspension containing the tagged protein was centrifuged again at 19,000 × g for 20 minutes at 4 ° C., and the supernatant was used as a lysate of the tagged protein.

  The lysate of the tagged protein was subjected to SDS-PAGE, the gel after electrophoresis was stained with Coomassie Brilliant Blue (CBB), and the color intensity of the band derived from the fusion protein was measured. Also, using a BSA solution containing bovine serum albumin (BSA) at a predetermined concentration, a calibration curve showing the color intensity of CBB with respect to the amount of protein was obtained. Based on this calibration curve, the amount of tagged protein contained in the solution of tagged protein was calculated, and the percentage of tagged protein re-solubilized after being recovered in the insolubilized fraction was examined.

  The results of this test example are shown in Table 8. In addition, "-" in a table | surface shows unexamined. As shown in Table 8, the fusion protein of mVenus and the insolubilizing tag could hardly be resolubilized when solvents 2, 11, 12 and 15 were used. Moreover, the fusion protein of IgHG3 and the insolubilizing tag was hardly able to be resolubilized when solvents 1 and 4 to 8 were used. On the other hand, when solvents 10, 18 and 23 were used, almost all of the fusion protein recovered in the insolubilized fraction was resolubilized for both fusion proteins.

  In addition, as shown in the solubilization rate in the solvents 16 to 20, when the solvent containing the concentration of SDS in the range of 0.04 to 1% (w / v) is used, the Venus recovered in the insolubilized fraction and The fusion protein with the insolubilizing tag was almost completely resolubilized (Table 8). Furthermore, as shown by the solubilization rate in solvents 21-24, in the solvent containing SDS at a concentration of 0.04% (w / v), the solubilization rate of the tagged protein is between pH 7.8 and pH 9.2. The fusion protein of IgHG3 and the insolubilizing tag recovered in the insolubilized fraction was almost completely resolubilized (Table 8).

<Test Example 4>
10. Resolubilization of Fusion Protein with Insolubilized Tag by Solvent The solvent 23 (see Table 7) examined in Test Example 3 is further suitable for resolubilization of a plurality of fusion proteins with insolubilized tag (tagged protein) I verified it.

  In the same manner as in Example 1, a fusion protein of a protein (see Table 9) and an insolubilization tag was synthesized using an entry clone into which the ORF sequence of the protein shown in Table 9 was cloned, and centrifuged to precipitate (insoluble fraction) Min) was obtained as a purified protein fraction. The solvent 23 was added to the purified protein fraction in the same manner as in Test Example 3 to obtain a solution of tagged protein. The quantification of the tagged protein contained in the solution was carried out in the same manner as in Test Example 3.

  The 28 types of proteins to which the insolubilization tag was added (Table 9) were almost completely resolubilized by the solvent 23, relative to the amount contained in the insolubilized fraction. From the results of this test example, after the tagged protein to which the protein tag according to the present invention is added is recovered in the insolubilized fraction, a solvent containing SDS at a concentration of 0.04% (w / v) is used. Confirmed that resolubilization is possible.

  According to the protein tag and the like according to the present invention, recombinant protein can be recovered easily with high yield, and comprehensive protein purification becomes possible. Therefore, the protein tag and the like according to the present invention can be used for basic research and clinical research using proteins, peptides, anti-protein antibodies and the like in the fields of medicine, medicine, biology and the like.

Claims (1)

Mass spectrometry of the target protein, which is a peptide mixture containing a tagged peptide obtained by trypsinizing a fusion protein of the target protein of mass spectrometry and a protein tag consisting of the amino acid sequence shown in SEQ ID NO: 4 derived from MafG protein Standard peptide.